Pharmaceutical compositions for combination therapy

ABSTRACT

The present invention relates to a combination product and its use in therapy.

The present invention relates to combinations of nitazoxanide (NTZ) or aderivative thereof in combination with a PPAR agonist for therapy.

[2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate, (ornitazoxanide, or NTZ), first described in 1975 (Rossignol and Cavier,1975), was shown to be highly effective against anaerobic protozoa,helminths, and a wide spectrum of microbes including both anaerobic andaerobic bacteria (Rossignol and Maisonneuve, 1984; Dubreuil, Houcke etal., 1996; Megraudd, Occhialini et al., 1998; Fox and Saravolatz, 2005;Pankuch and Appelbaum, 2006; Finegold, Molitoris et al., 2009). It wasfirst studied in humans for the treatment of intestinal cestodes(Rossignol and Maisonneuve, 1984) and it is now licensed in the UnitedStates (Alinia®, Romark laboratories) for the treatment of diarrheacaused by the protozoan parasites Crystosporidium parvum and Giardiaintestinalis. NTZ has also been widely commercialized in Latin Americaand in India where it is indicated for treating a broad spectrum ofintestinal parasitic infections (Hemphill, Mueller et al., 2006). Theproposed mechanism of action by which NTZ exerts its antiparasiticactivity is through the inhibition of pyruvate:ferredoxin oxidoreductase(PFOR) enzyme-dependent electron transfer reactions that are essentialfor anaerobic metabolism. (Hoffman, Sisson et al., 2007). NTZ alsoexhibited activity against Mycobacterium tuberculosis, which does notpossess a homolog of PFOR, thus suggesting an alternative mechanism ofaction. Indeed, the authors showed that NTZ can also act as an uncouplerdisrupting membrane potential and intra-organism pH homeostasis. (deCarvalho, Darby et al., 2011).

The pharmacological effects of NTZ are not restricted to itsantiparasitic or antibacterial activities and in recent years, severalstudies revealed that NTZ can also confer antiviral activity (Di Santoand Ehrisman, 2014; Rossignol, 2014). NTZ interferes with the viralreplication by diverse ways including a blockade in the maturation ofhemagglutinin (influenza) or VP7 (rotavirus) proteins, or the activationof the protein PKR involved in the innate immune response (for a review,see (Rossignol, 2014)). NTZ was also shown to have broad anticancerproperties by interfering with crucial metabolic and prodeath signalingpathways (Di Santo and Ehrisman, 2014).

The PPARs (α, β/δ (herein after δ), and γ), belong to thehormone-activated nuclear receptor family. The PPARs, or “PeroxisomeProliferator Activated Receptors”, are nuclear receptors from thesuperfamily of transcription factors activated by the following ligands:steroids/thyroid hormones/retinoids. To date, three PPAR isotypes havebeen identified in mice and humans: PPARα, PPARδ and PPARγ. WhilePPARβ/δ expression in humans appears to be ubiquitous, PPARα and γexhibit a differential tissue distribution (Braissant O and Wahli W,1998). PPARα is expressed in cells with high fatty acid catabolicactivity and in cells with high peroxisomal activity (hepatocytes,cardiomyocytes, renal proximal tubules, intestinal mucosa). PPARβ/δ isexpressed ubiquitously and abundantly in most tissues. As far as PPARγexpression is concerned, it is limited mainly to adipose tissue, certainimmune system cells and retina and is present in only trace amounts inother organs (Braissant O and Wahli W, 1998).

Taking the example of PPARα, its action is mediated by a class ofcompounds such as the fibrates which have a lipid-lowering effect.Natural ligands have also been identified such as for example fattyacids, eicosanoids (leukotriene B4) and 8(S)-hydroxyeicosatetraenoicacid (Kliewer S A et al., 1997). The PPARs have been associatedprimarily with lipid and glucose metabolism. PPAR activators, such asfibrates, enable a regulation of plasma cholesterol and triglycerideconcentrations via activation of PPARα (Hourton D et al., 2001). Fibratetherapy leads to an increase in fatty acid oxidation in liver. Fibratesalso decrease the synthesis of triglycerides (Staels B and Auwerx J,1998). PPARα activators are also capable of correcting hyperglycemia andinsulin level. Fibrates also reduce adipose tissue mass through amechanism which is independent of food intake and leptin gene expression(Guerre-Millo M et al., 2000). The therapeutic interest of PPARγagonists has been widely investigated in the treatment of type 2diabetes (Spiegelman B M, 1998). It has been shown that PPARγ agonistsrestore insulin sensitivity in target tissues and reduce plasma glucose,lipid and insulin levels both in animal models of type 2 diabetes and inhumans (Ram V J, 2003). PPAR activation by ligands also plays a role inregulating the expression of genes that participate in processes such asinflammation, angiogenesis, cell proliferation and differentiation,apoptosis and the activities of iNOS, MMPase and TIMPs. Activation ofPPARα a in keratinocytes results in a cessation of their proliferationand expression of genes involved in differentiation (Komuves L G et al.,2000). The PPARs have anti-inflammatory properties because theynegatively interfere with transcription mechanisms involving othertranscription factors like NF-κB or transcriptional activators like STATand AP-1 (Desvergne B and Wahli W, 1999). Said anti-inflammatory andanti-proliferative properties make the PPARs (and particularly PPARα)interesting therapeutic targets for the treatment of diseases such asvascular occlusive diseases (atherosclerosis, etc.), hypertension,diseases related to neovascularization (diabetic retinopathy, etc.),inflammatory diseases (inflammatory bowel disease, psoriasis, etc.) andneoplastic diseases (carcinogenesis, etc.)

The present invention describes novel combinations of NTZ analogues, andPPAR agonists and their use in therapy, in particular in the treatmentof inflammatory, metabolic, fibrotic and cholestatic diseases.

The inventors found that NTZ, a synthetic antiprotozoal agent or itsderivatives or its metabolites in combination with PPAR agonists showtherapeutic activities that are useful in therapy, in particular for thetreatment of immune, inflammatory, metabolic, fibrotic or cholestaticdiseases.

Therefore, the present invention relates to a combination productcomprising:

-   -   (i) a compound selected from NTZ and an analogue thereof; and    -   (ii) at least one PPAR agonist.

In a particular embodiment, the compound of component (i) of thecombination product is a compound of formula (I):

or a pharmaceutical acceptable salt thereof, in which:R1 represents a hydrogen atom (H), a deuterium atom (D), a halogen atom,a (C6-C14)aryl group, a heterocyclic group, a (C3-C14)cycloalkyl group,a (C1-C6)alkyl group, a sulfonyl group, a sulfoxyde group, a(C1-C6)alkylcarbonyl group, a (C1-C6)alkyloxy, a carboxylic group, acarboxylate group, a nitro group (NO2), an amino group (NH2), a(C1-C6)alkylamino group, an amido group, a (C1-C6)alkylamido group, a(C1-C6)dialkylamido group.R2 represents a hydrogen atom, a deuterium atom, a NO2 group, a(C6-C14)aryl group, a heterocyclic group, a halogen atom, a (C1-C6)alkylgroup, a (C3-C14)cycloalkyl group, a (C2-C6)alkynyl group, a(C1-C6)alkyloxy group, a (C1-C6)alkylthio group, a (C1-C6)alkylcarbonylgroup, a (C1-C6)alkylcarbonylamino group, a (C6-C14)arylcarbonylaminogroup, a carboxylic or carboxylate group, an amido group, a(C1-C6)alkylamido group, a (C1-C6)dialkylamido group, a NH2 group, a(C1-C6)alkylamino group,or R1 and R2, together with the carbon atoms to which they are attached,form a substituted or unsubstituted 5- to 8-membered cycloalkyl,heterocyclic or aryl group,R3, R4, R5, R6, and R7, identical or different, represent a hydrogenatom, a deuterium atom, a halogen atom, a hydroxyl group, a(C1-C6)alkylcarbonyl group, an (C1-C6)alkyl group, an (C1-C6)alkyloxygroup, an (C1-C6)alkylthio group, an (C1-C6)alkylcarbonyloxy group, an(C6-C14)aryloxy group, a (C6-C14)aryl group, a heterocyclic group, a(C3-C14)cycloalkyl group, a NO2 group, a sulfonylaminoalkyle group, anNH2 group, an amino(C1-C6)alkyl group, an (C1-C6)alkylcarbonylaminogroup, a carboxylic group, a carboxylate group, or a R9 group;R9 represents a O—R8 group or an amino-acid selected from the groupconsisting of alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, or a moiety of formula (A):

wherein R′ represents an (C1-C6)alkyl group, an (C2-C6)alkenyl group, an(C2-C6)alkynyl group, a (C3-C14)cycloalkyl group,(C3-C14)cycloalkylalkyl group, a (C3-C14)cycloalkyl(C2-C6)alkenyl group,a (C3-C14)cycloalkenyl group, a (C3-C14)cycloalkenyl(C1-C6)alkyl group,a (C3-C14)cycloalkenyl(C2-C6)alkenyl group, a(C3-C14)cycloalkenyl(C2-C6)alkynyl group; R″ and R′″, independently,represent hydrogen atom, an (C1-C6)alkyl group, or a nitrogen protectinggroup.R8 represents a hydrogen atom, a deuterium atom, a glucuronidyl group,or a

group wherein, R8a, R8b and R8c, identical or different, represent ahydrogen atom or a deuterium atom.

In a further particular embodiment, the compound of component (i) of thecombination product is a compound of formula (I):

or a pharmaceutical acceptable salt thereof, in which:R1 represents a hydrogen atom (H), a deuterium atom (D), a halogen atom,a (C6-C14)aryl group, a heterocyclic group, a (C3-C14)cycloalkyl group,a (C1-C6)alkyl group, a sulfonyl group, a sulfoxyde group, a(C1-C6)alkylcarbonyl group, a (C1-C6)alkyloxy, a carboxylic group, acarboxylate group, a NO2 group, a NH2 group, a (C1-C6)alkylamino group,an amido group, a (C1-C6)alkylamido group, a (C1-C6)dialkylamido group.R2 represents a hydrogen atom, a deuterium atom, a NO2 group, a(C6-C14)aryl group, a heterocyclic group, a halogen atom, a (C1-C6)alkylgroup, a (C3-C14)cycloalkyl group, a (C2-C6)alkynyl group, a(C1-C6)alkyloxy group, a (C1-C6)alkylthio group, a (C1-C6)alkylcarbonylgroup, a (C1-C6)alkylcarbonylamino group, a (C6-C14)arylcarbonylaminogroup, a carboxylic or carboxylate group, an amido group, a(C1-C6)alkylamido group, a (C1-C6)dialkylamido group, a NH2 group, a(C1-C6)alkylamino group, or R1 and R2, together with the carbon atoms towhich they are attached, form a substituted or unsubstituted 5- to8-membered cycloalkyl, heterocyclic or aryl group,R3 represents a hydrogen atom, a deuterium atom, a halogen atom, a O—R8group, a (C1-C6)alkylcarbonyl group, an (C1-C6)alkyl group, an(C1-C6)alkyloxy group, an (C1-C6)alkylthio group, an(C1-C6)alkylcarbonyloxy group, an (C6-C14)aryloxy group, a (C6-C14)arylgroup, a heterocyclic group, a (C3-C14)cycloalkyl group a NO2, asulfonylaminoalkyle group, an NH2 group, an amino(C1-C6)alkyl group, an(C1-C6)alkylcarbonylamino group, a carboxylic group, a carboxylategroup, an aminoacid selected from the group consisting of alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,or a moiety of formula (A):

wherein R′ represents an (C1-C6)alkyl group, an (C2-C6)alkenyl group, an(C2-C6)alkynyl group, a (C3-C14)cycloalkyl group,(C3-C14)cycloalkylalkyl group, a (C3-C14)cycloalkyl(C2-C6)alkenyl group,a (C3-C14)cycloalkenyl group, a (C3-C14)cycloalkenyl(C1-C6)alkyl group,a (C3-C14)cycloalkenyl(C2-C6)alkenyl group, a(C3-C14)cycloalkenyl(C2-C6)alkynyl group; R″ and R′″, independently,represent hydrogen atom, an (C1-C6)alkyl group, or a nitrogen protectinggroup.R8 represents a hydrogen atom, a deuterium atom, or a

group wherein, R8a, R8b and R8c, identical or different, represent ahydrogen atom or a deuterium atom. R4, R5, R6, and R7, identical ordifferent, represent a hydrogen atom, a deuterium atom, a halogen atom,a hydroxyl group, an (C1-C6)alkylcarbonyl group, an (C1-C6)alkyl group,an (C1-C6)alkyloxy group, an (C1-C6)alkylthio group, an(C1-C6)alkylcarbonyloxy group, an (C6-C14)aryloxy group, an (C6-C14)arylgroup, a heterocyclic group, a (C3-C14)cycloalkyl group, a NO2, asulfonylamino(C1-C6)alkyl group, an NH2 group, an amino(C1-C6)alkylgroup, an (C1-C6)alkylcarbonylamino group, a carboxylic group, acarboxylate group, an aminoacid selected from the group consisting ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, or a moiety of formula (A):

wherein R′ represents an (C1-C6)alkyl group, an (C2-C6)alkenyl group, an(C2-C6)alkynyl group, a (C3-C14)cycloalkyl group,(C3-C14)cycloalkyl(C1-C6)alkyl group, a (C3-C14)cycloalkyl(C1-C6)alkenylgroup, a (C3-C14)cycloalkenyl group, a (C3-C14)cycloakenyl(C1-C6)alkylgroup, a (C3-C14)cycloalkenyl(C2-C6)alkenyl group, a(C3-C14)cycloalkenyl(C2-C6)alkynyl group; R″ and R′″, independently,represent a hydrogen atom, an (C1-C6)alkyl group, or a nitrogenprotecting group;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, in the compound of formula (I) of thepresent invention:

an (C1-C6)alkyl group may be a substituted or unsubsituted (C1-C6)alkylgroup, in particular a substituted or unsubstituted (C1-C4)alkyl group;an (C2-C6)alkynyl group may be a substituted or unsubstituted(C2-C6)alkynyl group;a (C3-C14)cycloalkyl group may be a substituted or unsubstituted(C3-C14)cycloalkyl groupan (C1-C6)alkyloxy group may be either substituted or unsubstituted,such as a substituted or unsubstituted (C1-C4)alkyloxy group;an (C1-C6)alkylthio group may be either substituted or unsubstituted,such as a substituted or unsubstituted (C1-C4)alkylthio group;an (C1-C6)alkylamino group may be a (C1-C4)alkylamino group;a (C1-C6)dialkylamino group may be a (C1-C4)dialkylamino group;an (C6-C14)aryl group may be a substituted or unsubstituted (C6-C14)arylgroup;a heterocyclic group may be a substituted or unsubstitutedheterocycloalkyl or heteroaryl group.

In a particular embodiment, the compound of component (i) of thecombination product is a compound of formula (II)

in which R9 represents a hydrogen atom, a deuterium atom, a O—R8 group(R8 is defined above), or an aminoacid selected from the groupconsisting of alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, or a moiety of formula (A):

wherein R′ represents an (C1-C6)alkyl group, an (C2-C6)alkenyl group, an(C2-C6)alkynyl group, a (C3-C14)cycloalkyl group,(C3-C14)cycloalkyl(C1-C6)alkyl group, a (C3-C14)cycloalkyl(C1-C6)alkenylgroup, a (C3-C14)cycloalkenyl group, a (C3-C14)cycloakenyl(C1-C6)alkylgroup, a (C3-C14)cycloalkenyl(C2-C6)alkenyl group, a(C3-C14)cycloalkenyl(C2-C6)alkynyl group; R″ and R′″, independently,represent a hydrogen atom, an (C1-C6)alkyl group, or a nitrogenprotecting group or a pharmaceutically acceptable salt thereof.

In a particular embodiment, component (i) of the combination product ofthe invention is selected from:

In another embodiment, component (i) of the combination product is acompound of formula (I) or (II) wherein R8 represents a hydrogen atom, adeuterium atom or a

group wherein, R8a, R8b and R8c, identical or different, represent ahydrogen atom or a deuterium atom; and/or

-   -   wherein R1, R3, R4, R5, and R6, identical or different,        represent a hydrogen atom or a deuterium atom with the proviso        that R1, R2, R2a, R2b, R2c, R3, R4, R5, and R6 are not        simultaneously a hydrogen atom.

In a particular embodiment, component (i) is[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d3)ethanoate,2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d2) ethanoate; or2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d1) ethanoate.

In another particular embodiment, component (i) is((S)-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl2-amino-3,3-dimethylbutanoate hydrochloride) (RM5061) or formula:

In another particular embodiment, component (i) is((2S,3S)-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl2-amino-3-methylpentanoate hydrochloride) (RM5066) of formula:

In a further particular embodiment, the PPAR agonist is a PPAR-alphaagonist, a PPAR-gamma agonist, a PPAR-delta agonist, a PPAR-alpha/gammadual agonist, a PPAR alpha/delta dual agonist, a PPAR gamma/delta dualagonist or PPAR alpha/gamma/delta pan agonist.

According to the invention the PPAR agonist(s) and component (i)comprised in the combination of the invention may be selected so thatthe combination of said PPAR agonist(s) and component (i) of thecombination of the invention provides a synergistic action againstinflammatory, metabolic, fibrotic and cholestatic diseases. Such synergymay be determined according to methods well-known in the art, such as byusing the Excess Over Bliss (EOB) method described in the examples.

In a particular embodiment, component (ii) of the combination productis:

-   -   at least one PPAR-alpha agonist;    -   at least one PPAR-gamma agonist;    -   at least one PPAR-delta agonist;    -   at least one PPAR-alpha/delta dual agonist;    -   at least one PPAR-alpha agonist and at least one PPAR delta        agonist;    -   at least one PPAR-alpha/gamma dual agonist;    -   at least one PPAR-alpha agonist and at least one PPAR gamma        agonist;    -   at least one PPAR-gamma/delta dual agonist;    -   at least one PPAR-gamma agonist and at least one PPAR delta        agonist;    -   at least one PPAR-alpha/gamma/delta pan agonist; and    -   at least one PPAR-alpha agonist, at least one PPAR-gamma agonist        and at least one PPAR-delta agonist.

According to the present invention, the term “PPAR(s) agonists” refersthe Peroxisome Proliferator Activated Receptor agonists, which are aclass of drugs which plays a central role in lipid and glucosehomeostasis. PPARα mainly influences fatty acid metabolism and itsactivation lowers lipid levels, while PPARγ is mostly involved in theregulation of the adipogenesis, energy balance, and lipid biosynthesis.PPAR5 participates in fatty acid oxidation, mostly in skeletal andcardiac muscles, but it also regulates blood glucose and cholesterollevels.

According to the invention, the term “PPAR alpha agonist” as used hereinincludes, but is not limited to, fenofibrate, ciprofibrate, pemafibrate,gemfibrozil, clofibrate, binifibrate, clinofibrate, clofibric acid,nicofibrate, pirifibrate, plafibride, ronifibrate, theofibrate,tocofibrate, and SR10171.

According to the invention, the term “PPAR gamma agonist” as used hereinincludes, but is not limited to, Rosiglitazone, Pioglitazone, deuteratedpioglitazone, Efatutazone,

ATx08-001, OMS-405, CHS-131, THR-0921, SER-150-DN, KDT-501,GED-0507-34-Levo, CLC-3001, and ALL-4.

According to the invention, the term “PPAR delta agonist” as used hereinincludes, but is not limited to, GW501516 (Endurabol or({4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}aceticacid)), MBX8025 (Seladelpar or{2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[I,2,3]triazol-4-ylmethylsylfanyl]-phenoxy}-aceticacid), GW0742([4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]acetic acid), L165041, HPP-593, and NCP-1046.

According to the invention, the term “PPAR alpha/gamma agonist” (alsonamed glitazars) used herein includes, but is not limited toSaroglitazar, Aleglitazar, Muraglitazar, Tesaglitazar, and DSP-8658.

According to the invention, the term “PPAR alpha/delta agonist” usedherein includes, but is not limited to, Elafibranor (GFT505) or T913659.

According to the invention, the term “PPAR gamma/delta agonist” usedherein includes, but is not limited to a conjugated linoleic acid (CLA),T3D-959.

According to the invention, the term “PPAR alpha/gamma/delta agonist”used herein includes, but is not limited to, IVA337 (Lanifibranor), TTA(tetradecylthioacetic acid), Bavachinin, GW4148, GW9135, Bezafibrate,Lobeglitazone, and CS038. In a further embodiment, the PPARalpha/gamma/delta agonist is2-(4-(5,6-methylenedioxybenzo[d]thiazol-2-yl)-2-methylphenoxy)-2-methylpropanoicacid (MHY2013).

PPAR agonist may be in the form of a salt, hydrate, solvate, polymorph,or a co-crystal. PPAR agonist may also be in the form of a hydrate,solvate, polymorph, or a co-crystal of a salt.

In a more particular embodiment, the PPAR agonist is a compound offormula (III), or a pharmaceutically acceptable salt thereof:

in which:

Y1 represents a halogen, a Ra, or Ga-Ra group;

A represents a CH═CH or a CH2-CH2 group;

Y2 represents a Gb-Rb group;

Ga and Gb, identical or different, represent an atom of oxygen orsulfur;

Ra represents a hydrogen atom, an unsubstituted (C1-C6)alkyl group, a(C6-C14)aryl group or a (C1-C6)alkyl group that is substituted by one ormore halogen atoms, a (C1-C6)alkoxy or a (C1-C6)alkylthio group,(C3-C14)cycloalkyl groups, (C3-C14)cycloalkylthio groups or heterocyclicgroups;

Rb represents a (C1-C6)alkyl group substituted by at least a —COORcgroup, wherein

Rc represents a hydrogen atom, or a (C1-C6)alkyl group that issubstituted or not by one or more halogen atoms, (C3-C14)cycloalkylgroups, or heterocyclic groups; and

Y4 and Y5, identical or different, representing a (C1-C6)alkyl groupthat is substituted or not by one or more halogen atoms,(C3-C14)cycloalkyl groups or heterocyclic groups.

In a particular embodiment of the compound of formula (III):

Y1 represents a halogen, a Ra, or a Ga—Ra group;

A represents a CH═CH group;

Y2 represents a Gb-Rb group;

Ga and Gb, identical or different, represent an atom of oxygen orsulfur;

Ra represents a (C1-C6)alkyl or (C3-C14)cycloalkyl group, in particulara (C1-C7)alkyl or (C3-C14)cycloalkyl group substituted or not by one ormore halogen atoms;

Rb represents a (C1-C6)alkyl group substituted by a —COOR3 group,wherein Rc represents a hydrogen atom or an alkyl group having from oneto four carbon atoms; and

Y4 and Y5 independently represent a (C1-C4)alkyl group.

In a particular embodiment of the compound of formula (III):

Y1 represents a Ra or Ga—Ra group;

A represents a CH2-CH2 group;

Y2 represents a Gb-Rb group;

Ga represents an atom of oxygen or sulfur and Gb represents an atom ofoxygen;

Ra represents a (C1-C6)alkyl or (C3-C7)cycloalkyl group;

Rb represents a (C1-C6)alkyl group substituted by at least a —COORcgroup, wherein

Rc represents a hydrogen atom or (C1-C4)alkyl group; and

Y4 and Y5 independently represent a (C1-C4)alkyl group.

In a particular embodiment of the compound of formula (III):

Y1 represents a halogen atom or a Ra or Ga—Ra group;

A represents a CH2-CH2 group;

Y2 represents a Gb-Rb group;

Ga represents an atom of oxygen or sulfur and Gb represents an atom ofoxygen;

Ra represents a (C1-C6)alkyl or (C3-C14)cycloalkyl group that issubstituted by one or more halogen atoms;

Rb represents a (C1-C6)alkyl group substituted or not by one or morehalogen atoms and substituted by at least a —COORc group, wherein Rcrepresents a hydrogen atom or a (C1-C4)alkyl group; and

Y4 and Y5 represent a (C1-C4)alkyl group.

In a particular embodiment of the compound of formula (III), Gb is anoxygen atom and Rb is (C1-C6)alkyl group substituted by a —COORc group,wherein Rc represents a hydrogen atom or an unsubstituted linear orbranched (C1-C4)alkyl group.

In a particular embodiment of the compound of formula (III), Y1 is a(C1-C6)alkylthio group that comprises a (C1-C6)alkyl group that islinear or branched that is substituted or not by one or more halogenatoms.

In a particular embodiment, the compound of formula (III) is selected inthe group consisting of1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one (Elafibranor or GFT505),1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-isopropyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one,1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one,1-[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one,1-[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one,1-[4-trifluoromethyloxyphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one,1-[4-trifluoromethyloxyphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one,2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoicacid, and 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic acid isopropyl ester.

In a more particular embodiment, the PPAR agonist is1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one (or Elafibranor—GFT505), or a pharmaceuticallyacceptable salt thereof.

In a particular embodiment of the combination product of the invention:

-   -   component (ii) is selected in the group consisting of        Elafibranor, Saroglitazar, Seladelpar and Lanifibranor,        component (ii) being more particularly Elafibranor;    -   component (i) is selected from NTZ, TZ,        2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d3)ethanoate,        2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d2) ethanoate,        2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d1) ethanoate,        ((S)-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl        2-amino-3,3-dimethylbutanoate hydrochloride) or ((2S,3S)-2-(5-n        itroth iazol-2-ylcarbamoyl)phenyl 2-amino-3-methylpentanoate        hydrochloride).

In a further embodiment, component (i) is selected from TZG,2-(5-nitrothiazol-2-ylcarbamoyl)phenyl 2-amino-3,3-dimethylbutanoate(such as (S)-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl2-amino-3,3-dimethylbutanoate or its hydrochloride salt),2-(5-nitrothiazol-2-ylcarbamoyl)phenyl 2-amino-3-methylpentanoate (suchas (2S,3S)-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl2-amino-3-methylpentanoate or its hydrochloride salt),2-(5-chlorothiazol-2-ylcarbamoyl)phenyl 2-amino-3,3-dimethylbutanoate(such as (S)-2-(5-chlorothiazol-2-ylcarbamoyl)phenyl2-amino-3,3-dimethylbutanoate or its hydrochloride salt), and2-(5-chlorothiazol-2-ylcarbamoyl)phenyl 2-amino-3-methylpentanoate (suchas (2S,3S)-2-(5-chlorothiazol-2-ylcarbamoyl)phenyl2-amino-3-methylpentanoate or its hydrochloride salt). In a furthervariant of this embodiment, component (ii) is selected in the groupconsisting of Elafibranor, Saroglitazar Seladelpar and Lanifibranor,component (ii) being more particularly Elafibranor; and component (i) isselected from TZG, 2-(5-nitrothiazol-2-ylcarbamoyl)phenyl2-amino-3,3-dimethylbutanoate (such as(S)-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl 2-amino-3,3-dimethylbutanoateor its hydrochloride salt), 2-(5-nitrothiazol-2-ylcarbamoyl)phenyl2-amino-3-methylpentanoate (such as(2S,3S)-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl2-amino-3-methylpentanoate or its hydrochloride salt),2-(5-chlorothiazol-2-ylcarbamoyl)phenyl 2-amino-3,3-d imethylbutanoate(such as (S)-2-(5-chlorothiazol-2-ylcarbamoyl)phenyl2-amino-3,3-dimethylbutanoate or its hydrochloride salt), and2-(5-chlorothiazol-2-ylcarbamoyl)phenyl 2-amino-3-methylpentanoate (suchas (2S,3S)-2-(5-chlorothiazol-2-ylcarbamoyl)phenyl2-amino-3-methylpentanoate or its hydrochloride salt)

In a particular embodiment, the combination product of the inventionfurther comprises at least one other therapeutically active agent suchas an antifibrotic agent, an anti-inflammatory agent or animmunosuppressant agent.

Illustrative antifibrotic agents include pirfenidone or receptortyrosine kinase inhibitors (RTKIs) such as Nintedanib, Sorafenib andother RTKIs, or angiotensin II (AT1) receptor blockers, or CTGFinhibitor, or any antifibrotic compound susceptible to interfere withthe TGFβ and BMP-activated pathways including activators of the latentTGFβ complex such as MMP2, MMP9, THBS1 or cell-surface integrins, TGFβreceptors type I (TGFBRI) or type II (TGFBRII) and their ligands such asTGFβ, Activin, inhibin, Nodal, anti-Müllerian hormone, GDFs or BMPs,auxiliary co-receptors (also known as type III receptors), or componentsof the SMAD-dependent canonical pathway including regulatory orinhibitory SMAD proteins, or members of the SMAD-independent ornon-canonical pathways including various branches of MAPK signaling,TAK1, Rho-like GTPase signaling pathways, phosphatidylinositol-3kinase/AKT pathways, TGFβ-induced EMT process, or canonical andnon-canonical

Hedgehog signaling pathways including Hh ligands or target genes, or anymembers of the WNT, or Notch pathways which are susceptible to influenceTGFβ.

Illustrative anti-inflammatory and/or immunosuppressant agents includeglucocorticoids, NSAIDS, cyclophosphamide, nitrosoureas, folic acidanalogs, purine analogs, pyrimidine analogs, methotrexate, azathioprine,mercaptopurine, ciclosporin, myriocin, tacrolimus, sirolimus,mycophenolic acid derivatives, fingolimod and othersphingosine-1-phosphate receptor modulators, monoclonal and/orpolyclonal antibodies against such targets as proinflammatory cytokinesand proinflammatory cytokine receptors, T-cell receptor and integrins.

In a particular embodiment, the combination product of the invention isa composition comprising components (i) and (ii) as described above, anda pharmaceutically acceptable carrier.

In a particular embodiment, the combination product is a kit of partscomprising components (i) and (ii) as described above, for sequential,separate or simultaneous use.

In a further embodiment, components i) and ii) are formulated in aninjectable suspension, a gel, an oil, a pill, a tablet, a suppository, apowder, a capsule, an aerosol, an oinment, a cream, a patch, or means ofgalenic forms for a prolonged and/or slow release.

In another embodiment, components (i) and (ii) of the combinationproduct in the treatment of any of the diseases mentioned above.

The present invention also relates to the combination product accordingto the invention, for use as a medicament.

The invention also relates to the combination product herein disclosed,for use in a method for the treatment of the disease. In anotherembodiment, the invention relates to a method for the treatment of adisease, comprising administering to a subject in need thereof atherapeutically effective amount of the combination product hereindiscloses. In another embodiment, it is provided the use of acombination product according to the invention, for the manufacture of amedicament for the treatment of a disease.

In particular, the combination product of the present invention isuseful for the treatment of diseases such as immune, inflammatory,metabolic, fibrotic and cholestatic diseases. In a particularembodiment, the disease is selected in the group consisting of metabolicliver diseases, non-alcoholic fatty liver disease (NAFLD), non-alcoholicsteatohepatitis (NASH), drug-induced liver diseases, alcohol-inducedliver diseases, infectious agent induced liver diseases, inflammatoryliver diseases, immune system dysfunction-mediated liver diseases,dyslipidemia, cardiovascular diseases, restenosis, syndrome X, metabolicsyndrome, diabetes, obesity, hypertension, chronic cholangiopathies suchas Primary Sclerosing Cholangitis (PSC), Primary Biliary Cholangitis(PBC), biliary atresia, familial intrahepatic cholestasis type 3(PFIC3), inflammatory bowel diseases, Crohn's disease, ulcerativecolitis, keloid, old myocardial infarction, scleroderma/systemicsclerosis, inflammatory diseases, neurodegenerative diseases, cancers,liver cancer, hepatocallular carcinoma, gastrointestinal cancer, gastriccancer, meningioma associated with neurofibromatosis, pancreaticneuroendocrine tumors, pancreatic exocrine tumors, leukemia,myeloproliferative/myelodisplastic diseases, mastocytosis,dermatofibrosarcoma, solid tumors including breast, lung, thyroid orcolorectal cancer, a prostate cancer, liver fibrosis or cirrhosis of anyorigin, metabolic disease-induced liver fibrosis or cirrhosis,NAFLD-induced fibrosis or cirrhosis, NASH-induced fibrosis or cirrhosis,alcohol-induced liver fibrosis or cirrhosis, drug-induced liver fibrosisor cirrhosis, infectious agent-induced liver fibrosis or cirrhosis,parasite infection-induced liver fibrosis or cirrhosis, bacterialinfection-induced liver fibrosis or cirrhosis, viral infection-inducedfibrosis or cirrhosis, HBV-infection induced liver fibrosis orcirrhosis, HCV-infection induced liver fibrosis or cirrhosis,HIV-infection induced liver fibrosis or cirrhosis, dual HCV andHIV-infection induced liver fibrosis or cirrhosis, radiation- orchemotherapy-induced fibrosis or cirrhosis, biliary tract fibrosis,liver fibrosis or cirrhosis due to any chronic cholestatic disease, gutfibrosis of any etiology, Crohn's disease-induced fibrosis, ulcerativecolitis-induced fibrosis, intestine (e.g. small intestine) fibrosis,colon fibrosis, stomach fibrosis, skin fibrosis, epidermis fibrosis,endodermis fibrosis, skin fibrosis due to scleroderma/systemicsclerosis, lung fibrosis, lung fibrosis consecutive to chronicinflammatory airway diseases, such as COPD, asthma, emphysema, smoker'slung, tuberculosis, pulmonary fibrosis, idiopathic pulmonary fibrosis(IPF), heart fibrosis, kidney fibrosis, nephrogenic systemic fibrosis,muscle fibrosis, soft tissue (e.g. mediastinum or retroperitoneum)fibrosis, bone marrow fibrosis, joint fibrosis, tendon fibrosis,cartilage fibrosis, pancreas fibrosis, uterus fibrosis, nervous systemfibrosis, testis fibrosis, ovary fibrosis, adrenal gland fibrosis,artery fibrosis, vein fibrosis, eye fibrosis, endomyocardial fibrosis,mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis,progressive massive fibrosis (a complication of coal workers'pneumoconiosis), proliferative fibrosis, neoplastic fibrosis,peri-implantational fibrosis and asbestosis, arthrofibrosis, adhesivecapsulitis.

In a most preferred embodiment, the disease is selected in the groupconsisting of metabolic liver diseases, non-alcoholic fatty liverdisease (NAFLD), non-alcoholic steatohepatitis (NASH), drug-inducedliver diseases, alcohol-induced liver diseases, infectious agent inducedliver diseases, inflammatory liver diseases, immune systemdysfunction-mediated liver diseases, dyslipidemia, cardiovasculardiseases, restenosis, syndrome X, metabolic syndrome, diabetes, obesity,hypertension, chronic cholangiopathies such as Primary SclerosingCholangitis (PSC), Primary Biliary Cholangitis (PBC), biliary atresia,familial intrahepatic cholestasis type 3 (PFIC3), inflammatory boweldiseases, Crohn's disease, ulcerative colitis, liver cancer,hepatocallular carcinoma, gastrointestinal cancer, gastric cancer,colorectal cancer, metabolic disease-induced liver fibrosis orcirrhosis, NAFLD-induced fibrosis or cirrhosis, NASH-induced fibrosis orcirrhosis, alcohol-induced liver fibrosis or cirrhosis, drug-inducedliver fibrosis or cirrhosis, infectious agent-induced liver fibrosis orcirrhosis, parasite infection-induced liver fibrosis or cirrhosis,bacterial infection-induced liver fibrosis or cirrhosis, viralinfection-induced fibrosis or cirrhosis, HBV-infection induced liverfibrosis or cirrhosis, HCV-infection induced liver fibrosis orcirrhosis, HIV-infection induced liver fibrosis or cirrhosis, dual HCVand HIV-infection induced liver fibrosis or cirrhosis, radiation- orchemotherapy-induced fibrosis or cirrhosis, biliary tract fibrosis,liver fibrosis or cirrhosis due to any chronic cholestatic disease, gutfibrosis of any etiology, Crohn's disease-induced fibrosis, ulcerativecolitis-induced fibrosis, intestine (e.g. small intestine) fibrosis,colon fibrosis, stomach fibrosis, lung fibrosis, lung fibrosisconsecutive to chronic inflammatory airway diseases, such as COPD,asthma, emphysema, smoker's lung, tuberculosis, pulmonary fibrosis,idiopathic pulmonary fibrosis (IPF),

In a further aspect, the invention relates to the combination of theinvention, for use in the inhibition of proliferation and/or activationof fibroblasts responsible for the production of collagen fibers and/orresponsible for the production of the extracellular matrix.

According to the present invention, the term “autoimmune diseases” isused to designate a condition that arises from an abnormal immuneresponse of the body against substances and tissues normally present inthe body. The disease may be restricted to certain organs (e.g in type Idiabetes or autoimmune thyroiditis) or involve a particular tissue indifferent places (e.g. in Goodpasture's disease, affection of thebasement membrane in the lung and the kidney).

The term “inflammation” is used to designate a condition that arise froma protective response involving host cells, blood vessels, and proteinsand other mediators which may serve to eliminate the cause ofcell/tissue injury, as well as the necrotic cells/tissues resulting fromthe original insult, and to initiate the process of repair. Theinflammatory reaction may be manifested by pain, heat, redness,swelling, blood vessels dilatation, blood flow increase and loss offunction.

The terms “fibrosis”, “fibrotic disease”, “fibrotic disorder” anddeclinations thereof denote a pathological condition of excessivedeposition of fibrous connective tissue in an organ or tissue. Morespecifically, fibrosis is a pathologic process, which includes apersistent fibrotic scar formation and overproduction of extracellularmatrix, by the connective tissue, as a response to tissue damage.Physiologically, the deposit of connective tissue can obliterate thearchitecture and function of the underlying organ or tissue.

According to the present invention, the fibrosis or fibrotic disordermay be associated with any organ or tissue fibrosis. Illustrative,non-limiting examples of particular organ fibrosis include liver,kidney, skin, epidermis, endodermis, muscle, tendon, cartilage, heart,pancreas, lung, uterus, nervous system, testis, penis, ovary, adrenalgland, artery, vein, colon, intestine (e.g. small intestine), biliarytract, soft tissue (e.g. mediastinum or retroperitoneum), bone marrow,joint (e.g. knee, shoulder or other joints) or stomach fibrosis.

In a particular embodiment, the fibrotic disorder is selected in thegroup consisting of a liver, kidney, skin, epidermis, endodermis,muscle, tendon, cartilage, heart, pancreas, lung, uterus, nervoussystem, testis, ovary, adrenal gland, artery, vein, colon, intestine(e.g. small intestine), biliary tract, soft tissue (e.g. mediastinum orretroperitoneum), bone marrow, joint (e.g. knee, shoulder or otherjoints), eye or stomach fibrosis.

In a preferred embodiment, the fibrotic disorder is selected in thegroup consisting of a liver, gut, lung, heart, kidney, muscle, skin,soft tissue (e.g. mediastinum or retroperitoneum), bone marrow,intestinal, and joint (e.g. knee, shoulder or other joints) fibrosis.

In a more preferred embodiment, the fibrotic disorder is selected in thegroup consisting of the liver, lung, intestinal, heart, skin, kidney andintestinal fibrosis.

In another embodiment, the fibrotic disorder is selected in the groupconsisting of liver, lung, heart and intestinal fibrosis. In a moreparticular embodiment, the fibrotic disorder is selected in the groupconsisting of liver, lung, skin, kidney and intestinal fibrosis. Inanother embodiment, the fibrotic disorder is liver fibrosis.

In a more preferred embodiment of the present invention, treatedfibrotic disorder is selected in the group consisting of the followingnon exhaustive list of fibrotic disorders: non-alcoholic steatohepatitis(NASH), pulmonary fibrosis, idiopathic pulmonary fibrosis, skinfibrosis, eye fibrosis (such as capsular fibrosis), endomyocardialfibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis,progressive massive fibrosis (a complication of coal workers'pneumoconiosis), proliferative fibrosis, neoplastic fibrosis, lungfibrosis consecutive to chronic inflammatory airway disease (COPD,asthma, emphysema, smoker's lung, tuberculosis, IPF), alcohol ordrug-induced liver fibrosis, liver cirrhosis, infection-induced liverfibrosis, radiation or chemotherapeutic-induced fibrosis, nephrogenicsystemic fibrosis, Crohn's disease, ulcerative colitis, keloid, oldmyocardial infarction, scleroderma/systemic sclerosis, arthrofibrosis,some forms of adhesive capsulitis, chronic fibrosing cholangiopathiessuch as Primary Sclerosing Cholangitis (PSC) and Primary BiliaryCholangitis (PBC), biliary atresia, familial intrahepatic cholestasistype 3 (PFIC3), peri-implantational fibrosis and asbestosis.

Cholestasis is defined as a decrease in bile flow due to impairedsecretion by hepatocytes (hepato-cellular cholestasis) or to obstructionof bile flow through intra- or extrahepatic bile ducts (obstructivecholestasis). In clinical practice, cholestasis is any condition inwhich the flow of bile from the liver is slowed or blocked. According toa particular embodiment of the invention, the cholestestatic disease isselected in the group consisting of primary biliary cholangitis (PBC),primary sclerosing cholangitis (PSC), Intrahepatic Cholestasis ofPregnancy, Progressive Familial Intrahepatic Cholestasis, Biliaryatresia, Cholelithiasis, Infectious Cholangitis, Cholangitis associatedwith Langerhans cell histiocytosis, Alagille syndrome, Nonsyndromicductal paucity, Drug-induced cholestasis, and Total parenteralnutrition-associated cholestasis. In a preferred embodiment, thecholestatic disease is PBC or PSC, in particular PBC.

Examples of inflammatory diseases, fibrotic diseases, metabolic diseasesand cholestatic diseases include metabolic liver diseases, non-alcoholicfatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH),drug-induced liver diseases, alcohol-induced liver diseases, infectiousagent induced liver diseases, inflammatory liver diseases, immune systemdysfunction-mediated liver diseases, dyslipidemia, cardiovasculardiseases, restenosis, syndrome X, metabolic syndrome, diabetes, obesity,hypertension, chronic cholangiopathies such as Primary SclerosingCholangitis (PSC), Primary Biliary Cholangitis (PBC), biliary atresia,familial intrahepatic cholestasis type 3 (PFIC3), inflammatory boweldiseases, Crohn's disease, ulcerative colitis, keloid, old myocardialinfarction, scleroderma/systemic sclerosis, inflammatory diseases,neurodegenerative diseases, cancers, liver cancer, hepatocallularcarcinoma, gastrointestinal cancer, gastric cancer, meningiomaassociated with neurofibromatosis, pancreatic neuroendocrine tumors,pancreatic exocrine tumors, leukemia, myeloproliferative/myelodisplasticdiseases, mastocytosis, dermatofibrosarcoma, solid tumors includingbreast, lung, thyroid or colorectal cancer, a prostate cancer, liverfibrosis or cirrhosis of any origin, metabolic disease-induced liverfibrosis or cirrhosis, NAFLD-induced fibrosis or cirrhosis, NASH-inducedfibrosis or cirrhosis, alcohol-induced liver fibrosis or cirrhosis,drug-induced liver fibrosis or cirrhosis, infectious agent-induced liverfibrosis or cirrhosis, parasite infection-induced liver fibrosis orcirrhosis, bacterial infection-induced liver fibrosis or cirrhosis,viral infection-induced fibrosis or cirrhosis, HBV-infection inducedliver fibrosis or cirrhosis, HCV-infection induced liver fibrosis orcirrhosis, HIV-infection induced liver fibrosis or cirrhosis, dual HCVand HIV-infection induced liver fibrosis or cirrhosis, radiation- orchemotherapy-induced fibrosis or cirrhosis, biliary tract fibrosis,liver fibrosis or cirrhosis due to any chronic cholestatic disease, gutfibrosis of any etiology, Crohn's disease-induced fibrosis, ulcerativecolitis-induced fibrosis, intestine (e.g. small intestine) fibrosis,colon fibrosis, stomach fibrosis, skin fibrosis, epidermis fibrosis,endodermis fibrosis, skin fibrosis due to scleroderma/systemicsclerosis, lung fibrosis, lung fibrosis consecutive to chronicinflammatory airway diseases, such as COPD, asthma, emphysema, smoker'slung, tuberculosis, pulmonary fibrosis, idiopathic pulmonary fibrosis(IPF), heart fibrosis, kidney fibrosis, nephrogenic systemic fibrosis,muscle fibrosis, soft tissue (e.g. mediastinum or retroperitoneum)fibrosis, bone marrow fibrosis, joint fibrosis, tendon fibrosis,cartilage fibrosis, pancreas fibrosis, uterus fibrosis, nervous systemfibrosis, testis fibrosis, ovary fibrosis, adrenal gland fibrosis,artery fibrosis, vein fibrosis, eye fibrosis, endomyocardial fibrosis,mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis,progressive massive fibrosis (a complication of coal workers'pneumoconiosis), proliferative fibrosis, neoplastic fibrosis,peri-implantational fibrosis and asbestosis, arthrofibrosis, adhesivecapsulitis.

Preferably, the disease is selected in the group consisting of metabolicliver diseases, non-alcoholic fatty liver disease (NAFLD), non-alcoholicsteatohepatitis (NASH), drug-induced liver diseases, alcohol-inducedliver diseases, infectious agent induced liver diseases, inflammatoryliver diseases, immune system dysfunction-mediated liver diseases,dyslipidemia, cardiovascular diseases, restenosis, syndrome X, metabolicsyndrome, diabetes, obesity, hypertension, chronic cholangiopathies suchas Primary Sclerosing Cholangitis (PSC), Primary Biliary Cholangitis(PBC), biliary atresia, familial intrahepatic cholestasis type 3(PFIC3), inflammatory bowel diseases, Crohn's disease, ulcerativecolitis, liver cancer, hepatocallular carcinoma, gastrointestinalcancer, gastric cancer, colorectal cancer, metabolic disease-inducedliver fibrosis or cirrhosis, NAFLD-induced fibrosis or cirrhosis,NASH-induced fibrosis or cirrhosis, alcohol-induced liver fibrosis orcirrhosis, drug-induced liver fibrosis or cirrhosis, infectiousagent-induced liver fibrosis or cirrhosis, parasite infection-inducedliver fibrosis or cirrhosis, bacterial infection-induced liver fibrosisor cirrhosis, viral infection-induced fibrosis or cirrhosis,HBV-infection induced liver fibrosis or cirrhosis, HCV-infection inducedliver fibrosis or cirrhosis, HIV-infection induced liver fibrosis orcirrhosis, dual HCV and HIV-infection induced liver fibrosis orcirrhosis, radiation- or chemotherapy-induced fibrosis or cirrhosis,biliary tract fibrosis, liver fibrosis or cirrhosis due to any chroniccholestatic disease, gut fibrosis of any etiology, Crohn'sdisease-induced fibrosis, ulcerative colitis-induced fibrosis, intestine(e.g. small intestine) fibrosis, colon fibrosis, stomach fibrosis, lungfibrosis, lung fibrosis consecutive to chronic inflammatory airwaydiseases, such as COPD, asthma, emphysema, smoker's lung, tuberculosis,pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF).

The term “treatment” or “treating” refers to the curative or preventiveof a fibrotic disorder in a subject in need thereof. The treatmentinvolves the administration of the combination of the invention to asubject having a declared disorder, i.e. to a patient, to prevent, cure,delay, reverse, or slow down the progression of the disorder, improvingthereby the condition of the subject. A treatment may also beadministered to a subject that is healthy or at risk of developing afibrotic disorder.

Therefore, according to the invention, the treatment of a immune,inflammatory, metabolic, fibrotic and cholestatic disease involves theadministration of the combination of the present invention, for examplein the form of a pharmaceutical composition containing components (i)and (ii) of the combination, to a subject having a declared disorder tocure, delay, reverse or slow down the progression of the disorder, thusimproving the condition of the patient or to a healthy subject, inparticular a subject who is at risk of developing such disease.

The treatment involves the administration of the combination of theinvention to a patient having a declared disorder to cure, delay, orslow down the progress, thus improving the condition of the patient orto a healthy subject, in particular a subject who is at risk ofdeveloping an inflammatory, metabolic, fibrotic and cholestatic disease.

The subject to be treated is a mammal, preferably a human. The subjectto be treated according to the invention can be selected on the basis ofseveral criteria associated to fibrotic diseases such as previous drugtreatments, associated pathologies, genotype, exposure to risk factors,viral infection, as well as on the basis of the detection of anyrelevant biomarker that can be evaluated by means of imaging methods andimmunological, biochemical, enzymatic, chemical, or nucleic aciddetection methods.

The subjects to be treated according to the invention can be selected onthe basis of several criteria associated to inflammatory, metabolic,fibrotic and cholestatic diseases such as previous drug treatments,associated pathologies, genotype, exposure to risk factors, viralinfection, as well as any other relevant biomarker that can be evaluatedby means of imaging methods and immunological, biochemical, enzymatic,chemical, or nucleic acid detection method.

Synthesis of NTZ or analogues can be for example carried out asdescribed in (Rossignol and Cavier, 1975), or by any other way ofsynthesis known by a person skilled in the art.

In a particular embodiment, the treatment of an inflammatory, metabolic,fibrotic and cholestatic disease may comprise the administration of acomposition comprising at least two compounds selected from NTZ and NTZanalogues. In this embodiment, the administered at least one PPARagonist is provided in the same composition as the two compounds, or ina separate form, such as in a different composition.

In another embodiment, the combination of the invention is forsimultaneous, sequential or separate administration in therapy,therefore being possibly included in different compositions. In case ofsequential administration, NTZ and/or NTZ analogue(s) may beadministrated prior to the PPAR agonist(s), or the PPAR agonist(s) is(are) administrated prior to NTZ and/or NTZ analogue(s).

NTZ and NTZ analogue(s) can be formulated as pharmaceutically acceptablesalts, particularly acid or base salts compatible with pharmaceuticaluse. Salts of NTZ and NTZ analogue(s) include pharmaceuticallyacceptable acid addition salts, pharmaceutically acceptable baseaddition salts, pharmaceutically acceptable metal salts, ammonium andalkylated ammonium salts. These salts can be obtained during the finalpurification step of the compound or by incorporating the salt into thepreviously purified compound.

Combination of a compound of Formula (I) or (II) with one or more PPARagonist(s) can be formulated as pharmaceutically acceptable non-toxicsalts obtained from organic or inorganic bases or acids of compound ofFormula (I) or (II) or PPAR agonist(s). These salts can be obtainedduring the final purification step of the compound or by incorporatingthe salt into the previously purified compound.

The pharmaceutical compositions of the present invention comprising acompound of Formula (I) or (II) and one or more PPAR agonist(s) can alsocomprise one or several excipients or vehicles, acceptable within apharmaceutical context (e.g. saline solutions, physiological solutions,isotonic solutions, etc., compatible with pharmaceutical usage andwell-known by one of ordinary skill in the art).

These compositions can also comprise one or several agents or vehicleschosen among dispersants, solubilisers, stabilisers, preservatives, etc.Agents or vehicles useful for these formulations (liquid and/orinjectable and/or solid) are particularly methylcellulose,hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80,mannitol, gelatin, lactose, vegetable oils, acacia, liposomes, etc.

These compositions can be formulated in the form of injectablesuspensions, gels, oils, ointments, pills, suppositories, powders, gelcaps, capsules, aerosols, etc., eventually by means of galenic forms ordevices assuring a prolonged and/or slow release. For this kind offormulation, agents such as cellulose, carbonates or starches can beadvantageously used.

The pharmaceutical compositions of the present invention comprising acompound of Formula (I) or (II) and one or more PPAR agonist(s) may beadministered by different routes and in different forms. For example,the compound(s) may be administered via a systemic way, per os,parenterally, by inhalation, by nasal spray, by nasal instillation, orby injection, such as for example intravenously, by intra-muscularroute, by subcutaneous route, by transdermal route, by topical route, byintra-arterial route, etc.

Of course, the route of administration will be adapted to the form ofNTZ and/or NTZ analogue(s) in combination with one or more PPARagonist(s) according to procedures well known by those skilled in theart.

NTZ and/or NTZ analogue(s) in combination with one or more PPARagonist(s) is administered in a therapeutically effective amount. Withinthe context of the invention, the term “effective amount” refers to anamount of the compound sufficient to produce the desired therapeuticresult.

The frequency and/or dose relative to the administration can be adaptedby one of ordinary skill in the art, in function of the patient, thepathology, the form of administration, etc. Typically, the combination(such as in the form of a pharmaceutical composition or a kit-of-parts)of the present invention can be administered for the treatment of afibrotic disease at a dose for NTZ, or a NTZ analogue or a PPAR agonist,such as a compound of Formula (I) or (II) or (III), comprised between0.01 mg/day to 4000 mg/day, such as from 50 mg/day to 2000 mg/day, andparticularly from 100 mg/day to 1000 mg/day.

The dose of the PPAR agonist(s) in the said combination may varyaccording to the PPAR agonist itself. The dose is adapted to theefficiency of the PPAR agonist.

In a preferred embodiment of the invention, NTZ is used in combinationwith Elafibranor at a dose comprised between 100 mg/day and 1000 mg/day(such as 1000 mg/day) (in particular 20 mg/day to 40 mg/day) for NTZ and80 to 120 mg/day for Elafibranor.

In another preferred embodiment, the active ingredients are administeredas one or more pharmaceutical composition(s) in the form of a pillintended for an oral ingestion.

Administration can be performed daily or even several times per day, ifnecessary.

The invention is further described with reference to the following,non-limiting, examples.

DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1: Antifibrotic effect of Elafibranor and Nitazoxanide inTGFβ-induced hHSC

Serum-deprived HSC were preincubated for 1 hour with Elafibranor (A) orNitazoxanide (B), or Bezafibrate (C) before the activation with theprofibrogenic cytokine TGFI31 (1 ng/ml). After 48 hours of incubation,the expression of α-SMA was measured by ELISA. The obtained values weretransformed into percentage inhibition over TGFI31 control. Data arepresented as mean (triplicates)±standard deviation (SD)]. The curvefitting and the calculation of half maximal inhibitory concentration(IC₅₀) were performed with XLFit software 5.3.1.3.

FIG. 2: Combination of Elafibranor with Nitazoxanide synergisticallyinhibits α-SMA in TGFβ1-induced hHSC

Combinations were tested in a dose-response matrix format and analyzedaccording to the excess over Bliss (EOB) additivism model. Dilutionseries of Elafibranor (row) and Nitazoxanide (column) were prepared,including their respective DMSO controls. The resulting mixes were addedto serum-deprived HSC, 1 hour prior to the activation with theprofibrogenic cytokine TGFI31 (1 ng/ml). (A) Percentage of α-SMAinhibition over the TGFI31 control for all combination pairs. Data arepresented as mean of quadruplicates. (B) EOB scores were calculated asdescribed in Materials and Methods. Any compound pair with positive EOBvalue were considered synergistic (colored from light grey to black).The total EOB score including all combinations was also calculated. (C)Data values derived from a synergistic combination pair were plotted ina bar graph representation. Data are presented as mean(quadruplicates)±standard deviation (SD). EOHSA model was used asdescribed in the Materials and Methods section to confirm the synergismof the selected NTZ/ELA combination pairs.

FIG. 3: Combination of PPAR agonists with Nitazoxanide synergisticallyinhibits α-SMA in TGFβ1-induced hHSC

Combinations were tested in a dose-response matrix format and analyzedaccording to the excess over Bliss (EOB) additivism model. Dilutionseries of Saroglitazar, Seladelpar and Lanifibranor, and Nitazoxanidewere prepared, including their respective DMSO controls. The resultingmixes were added to serum-deprived HSC, 1 hour prior to the activationwith the profibrogenic cytokine TGFI31 (1 ng/ml). Percentage of α-SMAinhibition over the TGFI31 control for all combination pairs wascalculated. EOB scores were calculated as described in Materials andMethods. Any compound pair with positive EOB value were consideredsynergistic (colored from light grey to black).

(A) Sarogliazar (SARO)

(B) Seladelpar (SELA)

(C) Lanifibranor (LANI)

Percentage of α-SMA inhibition over the TGFI31 control derived from asynergistic combination pair were plotted in a bar graph representation.Data are presented as mean (quadruplicates)±standard deviation (SD).EOHSA model was used as described in the Materials and Methods sectionto confirm the synergism of the combination pairs.

FIG. 4: Hepatic collagen content

6 week-old C57BL/6 mice were fed a control (CSAA) diet, CDAA+1% CHOL(CDAAc) diet, or CDAAc diet supplemented with NTZ (30 mg/kg/day or 100mg/kg/day for 12 weeks) or Elafibranor (1 mg/kg/day or 3 mg/kg/day) or acombination of Elafibranor and NTZ (respectively 1+30 mg/kg/day, 1+100mg/kg/day, 3+30 mg/kg/day and 3+100 mg/kg/day). For each graph the exactamount of exposure doses was indicated.

After the sacrifice, the hepatic collagen content was determined.

FIG. 5: Hepatic fibrosis percentage

6 week-old C57BL/6 mice were fed a control (CSAA) diet, CDAA+1% CHOL(CDAAc) diet, or CDAAc diet supplemented with NTZ (30 mg/kg/day or 100mg/kg/day for 12 weeks) or Elafibranor (1 mg/kg/day or 3 mg/kg/day) or acombination of Elafibranor and NTZ (respectively 1+30 mg/kg/day, 1+100mg/kg/day, 3+30 mg/kg/day and 3+100 mg/kg/day). For each graph the exactamount of exposure doses was indicated.

After the sacrifice, the hepatic fibrosis area was determined.

FIG. 6: Hepatic αSMA gene expression

FIG. 7: Hepatic CCR2 gene expression

FIG. 8: Hepatic CCR5 gene expression

FIG. 9: Hepatic Col1a2 gene expression

FIG. 10: Hepatic MMP2 gene expression

FIG. 11: Hepatic TIMP2 gene expression

FIG. 12: Hepatic TGFβ1 gene expression

ABBREVIATIONS USED IN THE FIGURES, IN THE TABLES, AND IN THE TEXT

AP-1 activator protein 1ASBTi apical sodium-codependent bile acid transporter inhibitorASK1 signal-regulating kinase 1AT1 angiotensin 1

COPD Chronic Obstructive Pulmonary Disease

CTGF connective tissue growth factorDGAT diacylglycerol-O-acyltransferaseDMSO dimethylsulfoxydeDNA desoxyribonucleic acidDPP4 dipeptidyl peptidase

ELISA Enzyme-Linked Immuno Assay

EOB excess over BlissFABAC Fatty acid bile acid conjugateFBS fetal bovine serum

FGF Fibroblast Growth Factor

FXR Farnesoid X receptorGDF growth differentiation factorGLP-1 Glucagon-like peptide-1GPCR G-protein coupled receptorHBV hepatitis B virusHCV hepatitis C virus15-HEPE 5-hydroxyeicosapentaenoic acidHIV human immunodeficiency virusHSC hepatic stellate cellIC50 half maximal inhibitory concentrationiNOS inducible nitric oxide synthaseIPF idiopathic pulmonary fibrosis

LANI Lanifibranor

LBD ligand binding domainLPS lipopolysaccharideLT leukotrieneMAPK Mitogen-activated protein kinaseMMP-9 metalloprotease 9MMPase metalloproteaseNADPH nicotinamide adenine dinucleotide phosphateNAFLD non-alcoholic fatty liver diseaseNASH non-alcoholic steatohepatitisNF-κB nuclear factor-kappa BNOX NADPH oxidaseNSAIDs non-steroid anti-inflammatory drugs

NTZ Nitazoxanide

PAR Protease-activated receptor

PBC Primary Biliary Cholangitis

PDE phosphodiesterasePDGF platelet-derived growth factorPFIC3 familial intrahepatic cholestasis type 3PFOR pyruvate:ferredoxin oxidoreductasePPAR peroxisome proliferator activated receptor

PPRE PPAR Response Elements PSC Primary Sclerosing Cholangitis

ROCK Rho-associated protein kinaseRTK receptor tyrosine kinase

SARO Saroglitazar

SD standard deviation

SELA Seladelpar

SGLT Sodium-glucose transport

STAT Signal Transducers and Activators of Transcription

TGFβ transforming growth factor βTGFBRI TGFβ receptors type ITGFBRII TGFβ receptors type II

THBS1 Thrombospondin 1

THR β thyroid receptor βTIMP tissue inhibitor of metalloprotease

TLR-4 Toll Like Receptor 4 TZ Tizoxanide

TZG Tizoxanide glucuronideVAP-1 vascular adhesion protein-1

Examples Materials and Methods

Compounds were dissolved in dimethyl sulfoxide (DMSO, Fluka cat #41640).Nitazoxanide (INTERCHIM cat #RQ550U), tizoxanide (INTERCHIM cat #RP253),Lanifibranor (ARK PHARM cat #AK689102), Seladelpar (ARK PHARM cat#AK689146) and Saroglitazar (CHEMEXPRESS cat #YY-1997A) were obtainedcommercially.

hHSC Culture

The human primary hepatic stellate cells (hHSC) (Innoprot) were culturedin STeCM medium (ScienCell cat #5301) that was supplemented with 2%fetal bovine serum (FBS, ScienCell cat #0010), 1%penicillin/streptomycin (ScienCell cat #0503) and stellate cell growthsupplement (SteCGS; ScienCell cat #5352). Cell-culture flasks werecoated with Poly-L Lysine (Sigma cat #P4707) for a better adherence.

Preparation of Compositions 2 Components Combination Matrix(NTZ/Elafibranor)

For these experiments, a checkerboard matrix was generated. NTZ andElafibranor stocks were serially diluted in DMSO in 5-points series in arow (PPAR agonist Elafibranor) and a 6-points series in a column NTZ) ofa 96-well plate or a 384-well plate. Subsequently, the 5×6 combinationmatrix was generated by 1:1 mixing of all single agent concentrations.The test concentrations for each compound were chosen based on therespective IC₅₀ of each compound as single agent obtained by measuringα-SMA content in the HSC model stimulated with TGF-β1.

Then, 2-fold and 4-fold higher and lower concentrations were selected.

Activation of hHSC with TGF-β1 and Compound Treatment

The human primary hepatic stellate cells (hHSC) (Innoprot) were culturedunder standard conditions, as described above. The cells weresubsequently plated at a density of 2×10⁴ cells/well into 96-well platesand 6 500 cells/well into 384-well plates for the measure of α-SMA byELISA. The next day, cell-culture medium was removed, and cells werewashed with Phosphate-Buffered Saline (PBS) (Invitrogen cat #14190).hHSC were deprived for 24 hours in serum-free and SteCGS-free medium.

For the treatments with NTZ, Elafibranor or other PPAR agonists and therespective NTZ/Elafibranor or PPAR agonists combinations, theserum-deprived hHSC were preincubated for 1 hour with the compoundsfollowed by addition of the profibrogenic stimuli TGF-β1 (PeproTech cat#100-21, 1 ng/mL) in serum-free and SteCGS-free medium for an additional48 hour period.

α-SMA ELISA

The level of α-SMA was measured using a Sandwich ELISA. Briefly, thewells of an ELISA plate were first coated with the capture antibody(mouse monoclonal anti-ACTA2, Abnova) at 4° C. overnight. After 3 washesin PBS+0.2% Tween 20, a blocking solution consisting of PBS+0.2% BSA wasadded for one hour followed by another washing cycle. The cell lysateswere transferred into the wells for binding to the capture antibody fora period of 2 h at room temperature. After the washing procedure, thedetection antibody (biotinylated mouse monoclonal anti-ACTA2, Abnova)was added for 2 hours at room temperature followed by 3 washes. For thedetection, an HRP-conjugated Streptavidin (R&D Systems cat #DY998) wasfirst applied for 30 min at room temperature. After washing, the HRPsubstrate TMB (BD, #555214) was added and incubated for 7 min at roomtemperature in the dark. Upon oxidation, TMB forms a water-soluble bluereaction product that becomes yellow with addition of sulfuric acid(solution stop), enabling accurate measurement of the intensity at 450nm using a spectrophotometer. The developed color is directlyproportional to the amount of α-SMA present in the lysate.

Determination of Synergism by Excess Over Bliss (EOB) Method andConfirmation by EOSHA (Excess Over Highest Single Agent)

The values obtained in the αSMA ELISA assays were first transformed intopercentage inhibitions over TGF-β1 control. Then, using these percentageinhibitions, EOB (Excess Over Bliss) was determined to define thesynergistic effects of drug combinations. Expected Bliss additivismscore (E) was firstly determined by the equation:

E=(A+B)−(A×B) where A and B are the percentage inhibition of NTZ (A) andElafibranor, Saroglitazar, Seladelpar, or Lanifibranor (B) at a givendose. The difference between the Bliss expectation and the observedinhibition of the combined NTZ/Elafibranor, Saroglitazar, Seladelpar, orLanifibranor at the same dose is the ‘Excess over Bliss’ score.

-   -   Excess over Bliss score=0 indicates that the combination        treatment is additive (as expected for independent pathway        effects);    -   Excess over Bliss score >0 indicates activity greater than        additive (synergy); and    -   Excess over Bliss score <0 indicates the combination is less        than additive (antagonism).

For the combination NTZ+Elafibranor, an additional total Bliss score wascalculated by summation of all EOB.

EOHSA is a standard measure of synergy used by the FDA for evaluation ofdrug combinations and is calculated as the difference of the effectproduced by the drug combination and the greatest effect produced byeach of the combination's single agents at the same concentrations aswhen combined (Borisy et al., 2003). For synergistic combinationsidentified by the EOB method, the experimental % inhibition were plottedin a bar graph and the significance of the observed differences betweenNTZ/ELA and single agent were estimated by one-way ANOVA and uncorrectedFisher's LSD post-hoc (*: p<0.05; **: p<0.01; ***: p<0.001).

Evaluation of Elafibranor, Nitazoxanide and the CombinationElafibranor+Nitazoxanide in a Chronic CDAA+1% Cholesterol Model ifFibrosing NASH (12 Weeks)

Experimental Design

The choline-deficient and L-amino acid-defined (CDAA) diet lackscholine, which is essential for hepatic β-oxidation and very low densitylipoprotein production, and is believed to induce hepatocytes to storefat and subsequently cause cellular damage. The CDAA diet-induced rodentmodel develops fibrosis within a relatively short period of time, idealfor rapidly studying the reversibility of NASH pathology, particularlyfibrosis.

Increased cholesterol intake accelerates liver fibrosis in several mousemodels of NASH. The exacerbation of liver fibrosis mainly involves freecholesterol accumulation in hepatic stellate cells, that sensitizes thecells to transforming growth factor β (TGFβ) and subsequently aggravatesliver fibrosis.

In the current study, we examined the effects of nitazoxanide on liverfibrosis in C57Bl/6J mice fed a CDAA diet supplemented with 1%cholesterol.

The preventive effects of Elafibranor alone, NTZ alone and thecombination of both were assessed in a fibrosing NASH-model of mice feda CDAA+1% cholesterol diet. 6 week-old male C57Bl/6J mice were fed acontrol (CSAA) diet, CDAA+1% cholesterol diet, or CDAA+1% cholesteroldiet supplemented with Elafibranor 1, and 3 mg/kg/day, NTZ 30 and 100mg/kg/day or combined drugs (Elafibranor 1 and 3 mg/kg/day combined toNTZ 30 and 100 mg/kg/day) for 12 weeks.

The body weight and the food intake were monitored twice per week. Onthe last day of treatment, mice were sacrificed after a 6 h fastingperiod. The liver was rapidly excised for biochemical and histologicalstudies.

All animal procedures were performed according to standard protocols andin accordance with the standard recommendations for the proper care anduse of laboratory animals.

6 weeks-old C57BL/6 male mice were fed for 12 weeks according to anexperimental plan detailed in the table 1:

TABLE 1 Experimental plan Dose Diet Compound mg/kg/day Number of miceCSAA 8 CDAA + 1% Chol 12 Elafibranor 1 8 3 8 NTZ 30 8 100 8Elafibranor + 1 + 30  8 NTZ 1 + 100 8 3 + 30  8 3 + 100 8

The control was the CSAA diet.

Some mice were fed with the CDAAc diet.

Some mice were fed with CDAAc diet supplemented with Elafibranor at 1 or3 mg/kg/day.

Some mice were fed with CDAAc diet supplemented with NTZ at 30 or 100mg/kg/day.

Some mice were fed with CDAAc diet supplemented with Elafibranor+NTZcombination at different ratios: 1+30, 1+100, 3+30 and 3+100 mg/kg/day.

The group corresponding to CDAAc diet supplemented with NTZ at 30 mg/kgis also named C57Bl/6J mice fed CDAA diet+nitazoxanide 0.02% (wt/wt)corresponding to the theoretical dose of 30 mg/kg/day.

The group corresponding to CDAAc diet supplemented with NTZ at 100 mg/kgis also named C57Bl/6J mice fed CDAA diet+nitazoxanide 0.0667% (wt/wt)corresponding to the theoretical dose of 100 mg/kg/day.

The food was purchased from Ssniff® company (Soest, Germany).

Nitazoxanide (cf reference in Table 1) was incorporated by Ssniff® intoCDAA+1% chol diet in powder form to the required dose.

The reference and batch number of nitazoxanide are summarized in thetable Table 2:

TABLE 2 Nitazoxanide references Compound Laboratory code Refer- (INN)External ID Genfit ID Supplier ence Batch Nitazoxanide GFE 50455GSL022597.08 Interchim RQ550 1501

For each dose a calculation of the exact doses was done according to thefollowing example. This allows taking in count the exact dose of eachproduct that was exactly consumed by each group of mice.

Calculation of the Actual Treatment Doses: Example with Nitazoxanide0.02% (Wt/Wt)

-   -   Food intake is expressed in grams of food/grams of animal/day    -   0.02% of nitazoxanide in diet        -   0.02 g of cpd/100 g of food=0.2 mg of cpd/g of food    -   Actual dose of cpd:        -   0.2 mg of cpd/gram of food/gram of animal/day        -   (0.2 mg of cpd/gram of food/gram of animal/day)×1000=(0.2 mg            of cpd/gram of food/kg of animal/day)=200 mg of cpd/gram of            food/kg of animal per day

Consequently, multiplying by 200 the food intake value expressed ingrams of food/grams of animal/day; the obtained value corresponds to theactual administered dose expressed in mg of NTZ/kg of animal/day.

In the same manner:

-   -   For the dose of NTZ 0.00667% wt/wt, the actual treatment dose        was obtained by multiplying the food intake value (grams of        food/grams of animal/day) by 66.7.    -   For the dose of NTZ 0.0667% wt/wt, the actual treatment dose was        obtained by multiplying the food intake value (grams of        food/grams of animal/day) by 667.

According to the calculation the calculated dose versus estimated doseare given in the following tables 3 and 4.

TABLE 3 estimated and calculated doses for each compound a differentdoses Groups GFT505 NTZ Estimated doses 1mpk   3mpk   30mpk  100mpkCalculated doses 1mpk 2.8mpk 26.3mpk 78.1mpk

TABLE 4 estimated and calculated doses for each combinations atdifferent doses Combo 1 Combo 2 Combo 3 Combo 4 Groups GFT505 NTZ GFT505NTZ GFT505 NTZ GFT505 NTZ Estimated   1mpk   30mpk   1mpk  100mpk   3mpk  30mpk   3mpk  100mpk doses Calculated 0.9mpk 26.4mpk 0.9mpk 79.4mpk2.9mpk 27.4mpk 2.8mpk 77.7mpk doses

The body weight and food intake were recorded twice a week throughoutthe study.

At the end of the treatment period, animals were anesthetized withisoflurane and blood samples were taken as described below. Animals werethen sacrificed by cervical dislocation and beheaded for brain excisionand weighing. The liver was also collected and weighed. Part of theliver was fixed in 4% formalin, embedded in paraffin and used forhistological analyses. The remaining liver was snap frozen in liquidnitrogen and kept at −80° C. until use for further analyses.

Blood sampling was performed at sacrifice following a 6-hour fastingperiod. Blood samples were withdrawn under anesthesia by retro orbitalpuncture. Heparin tubes containing blood were rapidly centrifuged (15minutes at 4,000 rpm/4° C.) and the plasma fraction was collected.Plasma aliquots were stored at −20° C. until further analyses.

Plasma Biochemistry

Alanine Amino Transferase (ALT)

The plasmatic concentration of ALT was determined using the appropriateRandox kit for Daytona automate (Randox, cat #AL 3801). Briefly, the ALTwithin the plasma sample enzymatically transforms α-oxoglutarate andL-alanine into L-glutamate and pyruvate. In the presence of NADH, thegenerated pyruvate is converted by lactate dehydrogenase to formL-lactate and NAD⁺. The kinetics of the reaction is studied and allowsthe plasmatic level of ALT to be calculated. Results are expressed inU/L.

Aspartate Amino Transferase (AST)

The plasmatic concentration of AST was determined using the appropriateRandox kit for Daytona automate (Randox, cat #AS 3804). Briefly, the ASTwithin the plasma sample enzymatically transforms α-oxoglutarate andL-aspartate into L-glutamate and oxaloacetate. In the presence of NADH,the generated oxaloacetate is converted by malate dehydrogenase to formL-malate and NAD⁺. The kinetics of the reaction is studied and allowsthe plasmatic levels of AST to be calculated. Results are expressed inU/L.

Histology

At sacrifice, liver samples were processed for histological analysis andexamined as follows.

Tissue Embedding and Sectioning

The liver slices were first fixed for 12 hours in formalin 4% solution.Then, the liver pieces were washed 30 minutes in PBS, and dehydrated inethanol solutions (successive baths at 70, 80, 95 and 100% ethanol). Theliver pieces were incubated in three different baths of Xylene(Sigma-Aldrich cat #534056), followed by two baths in liquid paraffin(60° C.). Liver pieces were then put into racks that were gently filledwith Histowax® to completely cover the tissue.

The paraffin blocks containing the tissue pieces were removed from theracks and stored at room temperature. The liver blocks were cut into 3μm slices.

Picrosirius Red Staining

Liver sections were deparaffinized, rehydrated and incubated for 15minutes in a solution of Fast Green FCF 0.04% (Sigma-Aldrich, cat#F7258) before rinsing in a bath of 0.5% acetic acid (Sigma-Aldrich, cat#695092). Then, the liver sections were rinsed in water and incubated 30minutes in a solution of Fast Green FCF 0.04%-0.1% sirius red (DirectRed 80, Fluka cat #43665) in saturated aqueous picric acid(Sigma-Aldrich cat #P6744). Sections were then dehydrated, and mountedusing the CV Mount medium (Leica, cat #14046430011).

Histological Examinations

A technician blinded to the source of each liver specimen performedhistological examinations. Virtual slides were generated using thePannoramic 250 scanner from 3D Histech. Using Quant Center software (3DHistech, including Pattern Quant and Histo Quant modules),collagen-stained areas were quantified. Briefly, Pattern Quant was usedto detect the tissue and measure its surface. Then, Histo Quant was usedto detect the stained collagen content and measure its surface, based ona color threshold method. The fibrosis area was then expressed as thepercentage of the collagen surface to the whole tissue for each animal.

Hepatic fibrosis staging was blind-evaluated, using CRN fibrosiscriteria.

Details on the parameters, quantification/counting, and number of fieldsconsidered are provided in the following table 5.

TABLE 5 CRN's criteria for fibrosis Points Parameter in scoreDescription (the entire section was considered) Fibrosis 0 No fibrosis 1Centrilobular perisinusoidal/pericellular fibrosis or portal/periportalfibrosis 2 Centrilobular perisinusoidal/pericellular fibrosis andportal/periportal fibrosis 3 Centrilobular perisinusoidal/pericellularfibrosis and/or portal fibrosis with focal or extensive bridgingfibrosis 4 Cirrhosis

Statistical Analysis

Experimental results were expressed as mean±standard deviation (SD) andplotted as bar graphs or curves. Statistical analyses were performedusing Prism Version 7, as follows:

For the measures performed after sacrifice, CSAA vs CDAA+1% chol groupswere compared by a Student t-test (#: p<0.05; ##: p<0.01; ###: p<0.001)or by a Mann-Whitney test ($: p<0.05; $$: p<0.01; $$$: p<0.001).Treatment groups were compared to CDAA+1% chol diet by one-way ANOVA anduncorrected Fisher's LSD post-hoc (*: p<0.05; **: p<0.01; ***: p<0.001).

Measurement of Hepatic Collagen Content

The hepatic collagen content was determined using the appropriateQuickZyme kit (Total collagen assay, cat #QZB-totcol5). The assay isbased on the detection of hydroxyproline, which is a non-proteinogenicamino acid mainly found in the triple helix of collagen. Thus,hydroxyproline in tissue hydrolysates can be used as a direct measure ofthe amount of collagen present in the tissue (without discriminationbetween procollagen, mature collagen and collagen degradation products).

Complete hydrolysis of tissue samples in 6M HCl at 95° C. is requiredbefore dosing the hydroxyproline. The assay results in the generation ofa chromogen with a maximum absorbance at 570 nm. Results are expressedas mg of collagen/g of liver.

Procollagen III N-Terminal Propeptide (PIIINP)

The plasmatic concentration of PIIINP was determined using an ELISAassay from Cloud-Clone Corp (cat #SEA573Ra), according to themanufacturer's instructions. The microtiter plate is pre-coated with anantibody specific to PIIINP. Standards or samples are added to theappropriate microtiter plate wells with a biotin-conjugated antibodyspecific to PIIINP. Next, Avidin conjugated to Horseradish Peroxidase(HRP) is added to each microplate well and incubated. After TMBsubstrate solution is added, only those wells that contain PIIINP,biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit achange in color. The enzyme-substrate reaction is terminated by theaddition of sulphuric acid solution and the color change is measuredspectrophotometrically at a wavelength of 450 nm±10 nm. Theconcentration of PIIINP in the samples is then determined by comparingthe OD of the samples to the standard curve. Results are expressed inpg/mL.

Gene Expression

RNA Extraction

Hepatic Total RNA was isolated using Nucleospin® 96 Kit (Macherey Nagel)following manufacturer's instructions. 150 ng of total RNA were reversetranscribed in cDNA using M-MLV-RT (Moloney Murine Leukemia VirusReverse Transcriptase) (Invitrogen cat #28025) in presence of RT buffer1× (Invitrogen cat #P/NY02321), 1 mM DTT (Invitrogen cat #P/NY00147),0.5 mM dNTPs (Promega), 200 ng pdN6 (Roche cat #11034731001) and 40U ofRibonuclease inhibitor (Promega cat #N2515).

Quantitative PCR was then carried out using the CFX96 Touch™ Real-TimePCR Detection System (Biorad). Briefly, PCR reactions were performed in96 well plates on 5 μl of 5× diluted reverse transcription mix using theiQ SYBR Green Supermix kit (Biorad cat #170887). The experimentalconditions were: 20 μL of volume reaction and 0.5 μL each of reverse andforward primers (10 μMol).

Sequence Primer name ID Sequence (5′->3′) αSMA forward 1CTGACAGAGGCACCACTGAA αSMA reverse 2 CATCTCCAGAGTCCAGCACA Col1α1 forward3 AGGCGAACAAGGTGACAGAG Col1α1 reverse 4 GCCAGGAGAACCAGCAGAGCol1α2 forward 5 ATTGGAAGCCGAGGTCCCAG Col1α2 reverse 6TTTGCCCCCAGGTATGCCAG TGFβ1 forward 7 TTGCTTCAGCTCCACAGAGA TGFβ1 reverse8 TGGTTGTAGAGGGCAAGGAC TIMP1 forward 9 ATTCAAGGCTGTGGGAAATGTIMP1 reverse 10 CTCAGAGTACGCCAGGGAAC TIMP2 forward 11GCATCACCCAGAAGAAGAGC TIMP2 reverse 12 GGGTCCTCGATGTCAAGAAA MMP2 forward13 TCCCTAAGCTCATCGCAGAC MMP2 reverse 14 GCTTCCAAACTTCACGCTCTMMP7 forward 15 TAATTGGCTTCGCAAGGAGA MMP7 reverse 16AAGGCATGACCTAGAGTGTTCC CCR2 forward 17 TAATATGTTACCTCAGTTCATCCACGGCCR2 reverse 18 TGCTCTTCAGCTTTTTACAGCCTATC CCR5 forward 19ATTCTCCACACCCTGTTTCG CCR5 reverse 20 GAATTCCTGGAAGGTGGTCA GAP DH forward21 TATGACTCCACTCACGGCAA GAP DH reverse 22 TCCACGACATACTCAGCACC

Expression levels were normalized using the expression of GAPDH gene asreference.

For each gene, the standard curve was drawn by selecting the best points(at least three points) in order to have PCR reaction efficiency closeto 100% and a correlation coefficient close to 1. Expression levels weredetermined using the standard curve equation for both the housekeepinggene and the target gene (taking into account the specific PCRefficiency of each target gene).

Results and Conclusions:

The abnormal persistence of differentiated myofibroblasts is acharacteristic of many fibrotic diseases.

Following liver injury, quiescent HSCs undergo a process of activationthat is characterized by a differentiation into (α-SMA)-positivemyofibroblasts.

NTZ as a single agent was shown to confer an anti-fibrotic activity inTGFβ-induced hHSC (FIG. 1B). Since it is known that NTZ is rapidlyhydrolyzed into its active metabolite tizoxanide (TZ) (Broekhuysen,Stockis et al., 2000), this metabolite was also evaluated for itsantifibrotic activity in HSC. TZ showed a profile similar to the parentdrug (data not shown). On the other hand, some PPAR agonists likeElafibranor also revealed an antifibrotic profile in the TGFβ inducedHSC model (FIG. 1A). Other PPAR agonists like bezafibrate revealed aweak activity suggesting that PPAR agonists are not equivalent regardingtheir antifibrotic properties (FIG. 10).

In order to evaluate if a combination of Elafibranor with NTZ couldreduce fibrosis in a synergistic manner, combination matrix experimentswere performed in TGFβ-induced HSCs. Briefly, NTZ and Elafibranorsolutions were serially diluted in a checkerboard format generating a 42combinations matrix covering a large panel of Elafibranor/NTZ ratios.Synergy was first determined by calculating Excess Over Bliss scores.These experiments revealed that NTZ could synergize with Elafibranor toreduce α-SMA production in activated HSCs. Several combination pairsrevealed an EOB score over 10, which is indicative of a synergism (FIG.2B).

To validate the synergism, the experimental values corresponding to topEOB score were plotted in a bar graph (FIG. 2C). These graphs illustratethat the combination of NTZ with Elafibranor shows a superiorantifibrotic effect that is statistically significant compared to thehighest single agent (NTZ or Elafibranor). The most impressive exampleis represented with the combination pair NTZ at 0.6 μM and Elafibranorat 5 μM. Although NTZ shows almost no antifibrotic activity at 0.6 μM,the addition of Elafibranor at 5 μM results in a strong reduction ofαSMA of 55%, which is much stronger than the effect observed with thesingle agents alone. In order to evaluate if a combination of other PPARagonists, Sarogliatzar, Seladelpar and Lanifibranor with NTZ, couldreduce fibrosis in a synergistic manner, combination matrix experimentswere also performed in TGFβ-induced HSCs and EOB score were determined.To assess the synergism with the combination of NTZ with Saroglitazar,Seladelpar and Lanifibranor were validated on anti-fibrotic activity.The combination of NTZ with Saroglitazar, Seladelpar, or Lanifibranorrevealed an EOB score >0, which is indicative of a synergism. Thecombination of Sarogliatzar at 2.5 μM and NTZ at 0.625 μM results alsoin a strong reduction of αSMA of 52%, which is much stronger than theeffect observed with the single agents alone (FIG. 3A). The combinationof Seladelpar at 20 μM and NTZ at 2.5 μM also results in a strongreduction of αSMA of 72%, which is much stronger than the effectobserved with the single agents alone (FIG. 3B). To a lesser extent, thecombination of Lanifibranor at 10 μM and NTZ at 1.25 μM tends to besignificant (p=0.06) to reduce α-SMA production in activated HSCscompared to NTZ (FIG. 3C).

In conclusion, the applicant has discovered unexpected antifibroticactivities for a combination of a compound of Formula (I) with specificPPAR agonist(s). These results suggest that a combination of a compoundof Formula (I) with a PPAR agonist can be synergistic and can providetherapeutic benefits in multiple types of fibrotic diseases.

The administration of a choline-deficient and L-amino acid-deficient(CDAA)+1% cholesterol diet to mice causes progressive fibrosingsteatohepatitis that is pathologically similar to human non-alcoholicsteatohepatitis (NASH).

The CDAA+1% cholesterol diet notably induces a significant increase inthe hepatic collagen, as shown in the FIG. 4.

This figure also shows that administration of Elafibranor or NTZ alonedecreases the hepatic collagen content. The decrease in collagen isproportional to the dose of elafibranor or NTZ administered.

When a combination of Elafibranor and NTZ is administered, the decreasein collagen produced is greater than the decrease observed for eachcompound taken separately.

There is therefore a synergistic effect of the combination ofelafibranor and NTZ on the decrease in collagen production. In otherwords, there is a noticed anti-fibrotic effect when Elafibranor and NTZare combined.

The better effect is observed for the combination comprising Elafibranorat 2.8 mpk (mg per kilogram) and NTZ at 77.7 mpK, expressed incalculated doses.

FIG. 5 represents the results obtained with the histology, i.e. thedetermination of the fibrosis area which was expressed as the percentageof the collagen surface to the whole tissue for each animal.

The CDAA+1% cholesterol diet notably induces a significant increase inthe fibrosis percentage.

FIG. 5 also shows that administration of Elafibranor or NTZ alonedecreases the fibrosis percentage. The decrease is proportional to thedose of elafibranor or NTZ administered.

When a combination of Elafibranor and NTZ is administered, the decreasein fibrosis percentage is greater than the decrease observed for eachcompound taken separately.

There is therefore a synergistic effect of the combination ofelafibranor and NTZ on the decrease in the fibrosis percent. In otherwords, there is a noticed anti-fibrotic effect when Elafibranor and NTZare combined.

The better effect is observed for the combination comprising Elafibranorat 2.8 mpk (mg per kilogram) and NTZ at 77.7 mpK, expressed incalculated doses.

FIGS. 6 to 12 represent the gene expression of different hepatic markersof fibrosis. For all markers, the CDAA+1% cholesterol diet notablyinduces a significant increase in the gene expressions.

It can also be noted that that administration of Elafibranor or NTZalone decreases the level of the different gene expression. The decreaseis proportional to the dose of elafibranor or NTZ administered.

When a combination of Elafibranor and NTZ is administered, the decreasein gene expression is greater than the decrease observed for eachcompound taken separately.

There is therefore a synergistic effect of the combination ofelafibranor and NTZ on the decrease in the gene expression of differenthepatic markers of fibrosis. In other words, there is a noticedsynergistic anti-fibrotic effect when Elafibranor and NTZ are combined.

In conclusion, the applicant has discovered unexpected synergisticantifibrotic activities for a combination of a compound of Formula (I)with specific PPAR agonist(s). These results suggest that a combinationof a compound of Formula (I) with a PPAR agonist can be synergisticand/or can have additional effects and can also provide therapeuticbenefits in multiple types of fibrotic diseases.

REFERENCES

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1. A combination product comprising: (i) a compound selected from NTZand an analogue thereof; and (ii) at least one PPAR agonist.
 2. Thecombination product according to claim 1, wherein component (i) is acompound of formula (I):

or a pharmaceutical acceptable salt thereof, in which: R1 represents ahydrogen atom (H), a deuterium atom (D), a halogen atom, a (C6-C14)arylgroup, a heterocyclic group, a (C3-C14)cycloalkyl group, a (C1-C6)alkylgroup, a sulfonyl group, a sulfoxyde group, a (C1-C6)alkylcarbonylgroup, a (C1-C6)alkyloxy, a carboxylic group, a carboxylate group, a NO2group, a NH2 group, a (C1-C6)alkylamino group, an amido group, a(C1-C6)alkylamido group, a (C1-C6)dialkylamido group. R2 represents ahydrogen atom, a deuterium atom, a NO2 group, a (C6-C14)aryl group, aheterocyclic group, a halogen atom, a (C1-C6)alkyl group, a(C3-C14)cycloalkyl group, a (C2-C6)alkynyl group, a (C1-C6)alkyloxygroup, a (C1-C6)alkylthio group, a (C1-C6)alkylcarbonyl group, a(C1-C6)alkylcarbonylamino group, a (C6-C14)arylcarbonylamino group, acarboxylic or carboxylate group, an amido group, a (C1-C6)alkylamidogroup, a (C1-C6)dialkylamido group, a NH2 group, a (C1-C6)alkylaminogroup, or R1 and R2, together with the carbon atoms to which they areattached, form a substituted or unsubstituted 5- to 8-memberedcycloalkyl, heterocyclic or aryl group, R3 represents a hydrogen atom, adeuterium atom, a halogen atom, a O—R8 group, a (C1-C6)alkylcarbonylgroup, an (C1-C6)alkyl group, an (C1-C6)alkyloxy group, an(C1-C6)alkylthio group, an (C1-C6)alkylcarbonyloxy group, an(C6-C14)aryloxy group, a (C6-C14)aryl group, a heterocyclic group, a(C3-C14)cycloalkyl group a NO2, a sulfonylaminoalkyle group, an NH2group, an amino(C1-C6)alkyl group, an (C1-C6)alkylcarbonylamino group, acarboxylic group, a carboxylate group, an aminoacid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, or a moiety of formula (A):

wherein R′ represents an (C1-C6)alkyl group, an (C2-C6)alkenyl group, an(C2-C6)alkynyl group, a (C3-C14)cycloalkyl group,(C3-C14)cycloalkylalkyl group, a (C3-C14)cycloalkyl(C2-C6)alkenyl group,a (C3-C14)cycloalkenyl group, a (C3-C14)cycloalkenyl(C1-C6)alkyl group,a (C3-C14)cycloalkenyl(C2-C6)alkenyl group, a(C3-C14)cycloalkenyl(C2-C6)alkynyl group; R″ and R′″, independently,represent hydrogen atom, an (C1-C6)alkyl group, or a nitrogen protectinggroup. R8 represents a hydrogen atom, a deuterium atom, or a

 group wherein, R8a, R8b and R8c, identical or different, represent ahydrogen atom or a deuterium atom. R4, R5, R6, and R7, identical ordifferent, represent a hydrogen atom, a deuterium atom, a halogen atom,a hydroxyl group, an (C1-C6)alkylcarbonyl group, an (C1-C6)alkyl group,an (C1-C6)alkyloxy group, an (C1-C6)alkylthio group, an(C1-C6)alkylcarbonyloxy group, an (C6-C14)aryloxy group, an (C6-C14)arylgroup, a heterocyclic group, a (C3-C14)cycloalkyl group, a NO2, asulfonylamino(C1-C6)alkyl group, an NH2 group, an amino(C1-C6)alkylgroup, an (C1-C6)alkylcarbonylamino group, a carboxylic group, acarboxylate group, an aminoacid selected from the group consisting ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, or a moiety of formula (A):

wherein R′ represents an (C1-C6)alkyl group, an (C2-C6)alkenyl group, an(C2-C6)alkynyl group, a (C3-C14)cycloalkyl group,(C3-C14)cycloalkyl(C1-C6)alkyl group, a (C3-C14)cycloalkyl(C1-C6)alkenylgroup, a (C3-C14)cycloalkenyl group, a (C3-C14)cycloakenyl(C1-C6)alkylgroup, a (C3-C14)cycloalkenyl(C2-C6)alkenyl group, a(C3-C14)cycloalkenyl(C2-C6)alkynyl group; R″ and R′″, independently,represent a hydrogen atom, an (C1-C6)alkyl group, or a nitrogenprotecting group; or a pharmaceutically acceptable salt thereof.
 3. Thecombination production of claim 1 or 2, wherein component (i) is acompound of formula (II):

or a pharmaceutically acceptable salt thereof, in which R9 represents ahydrogen atom, a deuterium atom, a O—R8 group (R8 is defined above), oran aminoacid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine, or a moiety offormula (A):

wherein R′ represents an (C1-C6)alkyl group, an (C2-C6)alkenyl group, an(C2-C6)alkynyl group, a (C3-C14)cycloalkyl group,(C3-C14)cycloalkyl(C1-C6)alkyl group, a (C3-C14)cycloalkyl(C1-C6)alkenylgroup, a (C3-C14)cycloalkenyl group, a (C3-C14)cycloakenyl(C1-C6)alkylgroup, a (C3-C14)cycloalkenyl(C2-C6)alkenyl group, a(C3-C14)cycloalkenyl(C2-C6)alkynyl group; R″ and R′″, independently,represent a hydrogen atom, an (C1-C6)alkyl group, or a nitrogenprotecting group or a pharmaceutically acceptable salt thereof.
 4. Thecombination product according to any one of claims 1 to 3, whereincomponent (i) is selected from nitazoxanide, tizoxanide,[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d3)ethanoate,2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d2) ethanoate; or2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d1) ethanoate,((S)-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl2-amino-3,3-dimethylbutanoate hydrochloride) and ((2S,3S)-2-(5-n itrothiazol-2-ylcarbamoyl)phenyl 2-amino-3-methylpentanoate hydrochloride), orpharmaceutically acceptable salt thereof.
 5. The combination productaccording to any one of claims 1 to 4, wherein component (ii) isselected from: at least one PPAR-alpha agonist; at least one PPAR-gammaagonist; at least one PPAR-delta agonist; at least one PPAR-alpha/deltadual agonist; at least one PPAR-alpha agonist and at least one PPARdelta agonist; at least one PPAR-alpha/gamma dual agonist; at least onePPAR-alpha agonist and at least one PPAR gamma agonist; at least onePPAR-gamma/delta dual agonist; at least one PPAR-gamma agonist and atleast one PPAR delta agonist; at least one PPAR-alpha/gamma/delta panagonist; and at least one PPAR-alpha agonist, at least one PPAR-gammaagonist and at least one PPAR-delta agonist.
 6. The combination productaccording to any one of claims 1 to 5, wherein the at least one PPARagonist is a compound of formula (III), or a pharmaceutically acceptablesalt thereof:

in which: Y1 represents a halogen, a Ra, or Ga—Ra group; A represents aCH═CH or a CH2-CH2 group; Y2 represents a Gb-Rb group; Ga and Gb,identical or different, represent an atom of oxygen or sulfur; Rarepresents a hydrogen atom, an unsubstituted (C1-C6)alkyl group, a(C6-C14)aryl group or a (C1-C6)alkyl group that is substituted by one ormore halogen atoms, a (C1-C6)alkoxy or a (C1-C6)alkylthio group,(C3-C14)cycloalkyl groups, (C3-C14)cycloalkylthio groups or heterocyclicgroups; Rb represents a (C1-C6)alkyl group substituted by at least a—COORc group, wherein Rc represents a hydrogen atom, or a (C1-C6)alkylgroup that is substituted or not by one or more halogen atoms,(C3-C14)cycloalkyl groups, or heterocyclic groups; and Y4 and Y5,identical or different, representing a (C1-C6)alkyl group that issubstituted or not by one or more halogen atoms, (C3-C14)cycloalkylgroups or heterocyclic groups.
 7. The combination product according toany one of claims 1 to 6, wherein component (ii) is selected fromElafibranor, Seladelpar, Saroglitazar, and Lanifibranor, or apharmaceutical salt thereof.
 8. The combination product according to anyone of claims 1 to 7, comprising: (i) a compound selected fromnitazoxanide, tizoxanide, [(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl(d3)ethanoate, 2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d2)ethanoate; or 2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl (d1)ethanoate, ((S)-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl2-amino-3,3-dimethylbutanoate hydrochloride) and((2S,3S)-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl2-amino-3-methylpentanoate hydrochloride); or from a pharmaceutical saltthereof; and (ii) Elafibranor or a pharmaceutically acceptable saltthereof.
 9. The combination product according to any one of claims 1 to8, wherein the combination product is a composition comprisingcomponents i) and ii) and a pharmaceutically acceptable carrier.
 10. Thecombination product according to any one of claims 1 to 9, wherein thecombination product is a kit of parts comprising components i) and ii),for sequential, separate or simultaneous use.
 11. The combinationproduct according to any one of claims 1 to 10, further comprising atleast one therapeutically active agent with known antifibrotic activityselected from pirfenidone or receptor tyrosine kinase inhibitors (RTKIs)such as Nintedanib, Sorafenib and other RTKIs, or angiotensin II (AT1)receptor blockers, or CTGF inhibitor, or any antifibrotic compoundsusceptible to interfere with the TGFβ and BMP-activated pathwaysincluding activators of the latent TGFβ complex such as MMP2, MMP9,THBS1 or cell-surface integrins, TGFβ receptors type I (TGFBRI) or typeII (TGFBRII) and their ligands such as TGFβ, Activin, inhibin, Nodal,anti-Müllerian hormone, GDFs or BMPs, auxiliary co-receptors (also knownas type III receptors), or components of the SMAD-dependent canonicalpathway including regulatory or inhibitory SMAD proteins, or members ofthe SMAD-independent or non-canonical pathways including variousbranches of MAPK signaling, TAK1, Rho-like GTPase signaling pathways,phosphatidylinositol-3 kinase/AKT pathways, TGFβ-induced EMT process, orcanonical and non-canonical Hedgehog signaling pathways including Hhligands or target genes, or any members of the WNT, or Notch pathwayswhich are susceptible to influence TGFβ.
 12. The combination productaccording to any one of claims 1 to 11, further comprising at least onetherapeutically active agent selected from JAK/STAT inhibitors and otheranti-inflammatory and/or immunosuppressant agents.
 13. The combinationproduct according to claim 12, wherein the therapeutically active agentis selected from glucocorticoids, NSAIDS, cyclophosphamide,nitrosoureas, folic acid analogs, purine analogs, pyrimidine analogs,methotrexate, azathioprine, mercaptopurine, ciclosporin, myriocin,tacrolimus, sirolimus, mycophenolic acid derivatives, fingolimod andother sphingosine-1-phosphate receptor modulators, monoclonal and/orpolyclonal antibodies against such targets as proinflammatory cytokinesand proinflammatory cytokine receptors, T-cell receptor and integrins.14. The combination product according to any one of claims 1 to 13,wherein components (i) and (ii) are formulated in an injectablesuspension, a gel, an oil, a pill, a tablet, a suppository, a powder, acapsule, an aerosol, an oinment, a cream, a patch, or means of galenicforms for a prolonged and/or slow release.
 15. The combination productaccording to any one of claims 1 to 14, for use as a medicament.
 16. Thecombination product according to any one of claims 1 to 14, for use in amethod for treating an inflammatory, metabolic, fibrotic and cholestaticdisease.
 17. The combination product for use according to claim 16,wherein the fibrotic disorder is selected in the group consisting ofliver, kidney, skin, epidermis, endodermis, muscle, tendon, cartilage,heart, pancreas, lung, uterus, nervous system, testis, ovary, adrenalgland, artery, vein, colon, intestine (e.g. small intestine), biliarytract, soft tissue (e.g. mediastinum or retroperitoneum), bone marrow,joint and stomach fibrosis, in particular liver, gut, lung, heart,kidney, muscle, skin, soft tissue, bone marrow, intestinal, and jointfibrosis.
 18. The combination product for use according to claim 16,wherein the disease is selected in the group consisting of, metabolicliver diseases, non-alcoholic fatty liver disease (NAFLD), non-alcoholicsteatohepatitis (NASH), drug-induced liver diseases, alcohol-inducedliver diseases, infectious agent induced liver diseases, inflammatoryliver diseases, immune system dysfunction-mediated liver diseases,dyslipidemia, cardiovascular diseases, restenosis, syndrome X, metabolicsyndrome, diabetes, obesity, hypertension, chronic cholangiopathies suchas Primary Sclerosing Cholangitis (PSC) and Primary Biliary Cholangitis(PBC).